Vaccines and methods to treat canine influenza

ABSTRACT

The present invention relates to providing new vaccines and treatments for the diseases related to canine influenza virus. It discloses influenza viral antigens, and methods of presenting these antigens to canines, especially dogs. It relates to attenuated and killed vaccines. The present invention relates to experimentally generated canine and equine influenza viruses. invention also includes influenza A, including H3, N8, H3N8, H7N7 and viruses which contain at least one genome segment from an canine or equine influenza virus. The present invention also relates to the use of these viruses in therapeutic compositions to protect canines, dogs in particular, from diseases caused by influenza viruses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/544,841, filed Oct. 6, 2006, now U.S. Pat. No. 7,722,884, whichclaims the benefit of U.S. Provisional Patent Application No.60/724,827, filed Oct. 7, 2005, the entire disclosures of bothapplications are incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to providing new vaccines and treatmentsfor the diseases related to canine influenza virus. It disclosesinfluenza viral antigens, and methods of presenting these antigens tocanines, especially dogs. It relates to attenuated and killed vaccines.

The present invention relates to experimentally generated canine andequine influenza vaccines and viruses. The invention also includesinfluenza A, H3, N8, H3N8, and H7N7 viruses which contain at least onegenome segment from a canine or equine influenza virus. The presentinvention also relates to the use of these viruses in therapeuticcompositions to protect canines, dogs in particular, from diseasescaused by influenza viruses.

BACKGROUND OF THE INVENTION

Equine influenza virus has been recognized as a major respiratorypathogen in horses since about 1956. Disease symptoms caused by equineinfluenza virus can be severe, and are often followed by secondarybacterial infections. Two subtypes of equine influenza virus arerecognized, namely subtype-1, the prototype being A/Equine/Prague/1/56(H7N7), and subtype-2, the prototype being A/Equine/Miami/1/63 (H3N8).Presently, the predominant virus subtype is subtype-2, the H3N8 strain.It is now believed that this strain may be infecting canines and it canbe quite virulent with canine fatality rates reported in some cases ashigh as 36%. It is possible that an interspecies transfer of thecomplete or a portion of the equine influenza virus to the dog resultedin a new canine specific influenza virus associated with acuterespiratory disease. See, Transmission of Equine Influenza to Dogs (P.C. Crawford et al., Science 310, 482-485 (2005). There is a clear andconvincing need for an effective vaccine to treat and prevent this newcanine influenza.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows geometric mean injection site reactions of dogs vaccinatedwith Equine Antigen Vaccines.

FIG. 2 shows mean percent lung consolidation in animals vaccinated withEquine Influenza Virus Vaccines and challenged with Canine InfluenzaVirus.

SUMMARY OF THE INVENTION

The present invention provides for equine and canine influenza antigens,vaccines, and methods of using those vaccines to treat canines,especially dogs, from infections, disease and symptoms caused by canineinfluenza. The invention further provides therapeutic compositions toprotect an animal against disease caused by influenza virus. Methods ofmaking the vaccines and methods of treating animals are describedherein. The antigens of this invention may be any identified influenzavirus strain, from any bird or mammal, including but not limited toinfluenza having the H3N8 antigenic subtype or more commonly referred toas an H3N8 strain. The influenza may be of any mammalian origin,including but not limited to swine, avian, equine or canine origin.Equine and Canine influenza virus and related antigens are preferred.Strains having the proteins designated H3 or N8 are disclosed. Stainshaving both H3N8 are preferred. Strains having the proteins designatedH7N7 are also disclosed.

Antigen concentration and vaccine production are described. Cell culturemedia and viral growth is described. Vaccine preparation of attenuated,killed and inactivated virus as well as vaccine adjuvants, formulations,forms, and carriers, dosages, routes of administration and assays areall described.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Abbreviations

The definitions below apply to this disclosure, words not defined havethe meaning commonly used by one skilled in the art.

“About,” when used in connection with a measurable numerical variable,refers to the indicated value of the variable and to all values of thevariable that are within the experimental error of the indicated value(e.g., within the 95% confidence interval for the mean) or within 10percent of the indicated value, whichever is greater.

“Active immunity” includes both humoral immunity and/or cell-mediatedimmunity in a dog.

“Antibody” refers to an immunoglobulin molecule that can bind to aspecific antigen as the result of an immune response to that antigen.Immunoglobulins are serum proteins composed of “light” and “heavy”polypeptide chains having “constant” and “variable” regions and aredivided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on thecomposition of the constant regions. An antibody that is “specific” fora given antigen indicates that the variable regions of the antibodyrecognize and bind a specific antigen exclusively. Antibodies can be apolyclonal mixture or monoclonal. Antibodies can be intactimmunoglobulins derived from natural sources or from recombinantsources, or can be immunoreactive portions of intact immunoglobulins.Antibodies can exist in a variety of forms including, for example, as,Fv, Fab′, F(ab′)₂, as well as in single chains.

“Antigen” or “immunogen” refers to a molecule that contains one or moreepitopes (linear, conformational or both) that upon exposure to asubject will induce an immune response that is specific for thatantigen. An epitope is the specific site of the antigen which binds to aT-cell receptor or specific antibody, and typically comprises about 3amino acid residues to about 20 amino acid residues. The term antigenrefers to subunit antigens—antigens separate and discrete from a wholeorganism with which the antigen is associated in nature—as well askilled, attenuated or inactivated bacteria, viruses, fungi, parasites orother microbes. The term antigen also refers to antibodies, such asanti-idiotype antibodies or fragments thereof, and to synthetic peptidemimotopes that can mimic an antigen or antigenic determinant (epitope).The term antigen also refers to an oligonucleotide or polynucleotidethat expresses an antigen or antigenic determinant in vivo, such as inDNA immunization applications.

“Antigenicity” refers to the capability of a protein or polypeptide tobe immunospecifically bound by an antibody raised against the protein orpolypeptide.

“Canine” includes what is commonly called the dog, but includes othermembers of the family Canidae.

“Cellular Immune Response”—see Immune Response.

“Companion animal”, as used herein, refers to any non-human animal incaptivity considered to be a pet. These may include, but are notrestricted to, dogs, cats, horses, sheep, rabbits, monkeys, and rodents,including mice, rats, hamsters, gerbils, and ferrets.

“Equine” includes what is commonly called the horse, but includes othermembers of the family Equidae

“Excipient” refers to any component of a vaccine that is not an antigen.

“First vaccine,” “second vaccine,” “third vaccine,” and the like, referto separately administrable vaccines, which may be the same ordifferent, and which in general may be administered in any order. Thus,a third vaccine may be administered to a subject before or after asecond vaccine.

“Heterologous”, when used herein means derived from a different viral,species or strain.

“Homology”, “homologous”, and the like, when used herein means thedegree of identity shared between polynucleotide or polypeptidesequences.

“Homologous”, when used in reference to a viral, species means the sameviral species or strain.

“Host cell”, when used herein means a bacteria or eukaryotic cell,including mammalian, avian or insect, that harbors a plasmid, virus, orother vector.

“Humoral Immune Response”—see Immune Response.

“Hybridoma”—see Monoclonal Antibody.

“Immune response” in a subject refers to the development of a humoralimmune response, a cellular immune response, or a humoral and a cellularimmune response to an antigen. A “humoral immune response” refers to onethat is mediated by antibodies. A “cellular immune response” is onemediated by T-lymphocytes or other white blood cells or both, andincludes the production of cytokines, chemokines and similar moleculesproduced by activated T-cells, white blood cells, or both. Immuneresponses can be determined using standard immunoassays andneutralization assays, which are known in the art.

“Immunogenicity” refers to the capability of a protein or polypeptide toelicit an immune response directed specifically against a bacteria orvirus that causes the identified disease.

“Immunologically protective amount” or “effective amount to produce animmune response” of an antigen is an amount effective to induce animmunogenic response in the recipient that is adequate to prevent orameliorate signs or symptoms of disease, including adverse healtheffects or complications thereof. Either humoral immunity orcell-mediated immunity or both may be induced. The immunogenic responseof an animal to a vaccine composition may be evaluated, e.g., indirectlythrough measurement of antibody titers, lymphocyte proliferation assays,or directly through monitoring signs and symptoms after challenge withwild type strain. The protective immunity conferred by a vaccine can beevaluated by measuring, e.g., reduction in clinical signs such asmortality, morbidity, temperature number and overall physical conditionand overall health and performance of the subject. The immune responsemay comprise, without limitation, induction of cellular and/or humoralimmunity. The amount of a vaccine that is therapeutically effective mayvary depending on the particular virus used, or the condition of theanimal being vaccinated, and can be determined by a veterinaryphysician.

“Intranasal” administration refers to the introduction of a substance,such as a vaccine, into a subject's body through or by way of the noseand involves transport of the substance primarily through the nasalmucosa.

“Isolated” when used herein means removed from its naturally occurringenvironment, either alone or in a heterologous host cell, or chromosomeor vector (e.g., plasmid, phage, etc.). “Isolated bacteria,” “isolatedanaerobic bacteria,” “isolated bacterial strain,” “isolated virus”“isolated viral strain” and the like refer to a composition in which thebacteria or virus are substantial free of other microorganisms, e.g., ina culture, such as when separated from it naturally occurringenvironment. “Isolated,” when used to describe any particularly definedsubstance, such as a polynucleotide or a polypeptide, refers to thesubstance that is separate from the original cellular environment inwhich the substance such as a polypeptide or nucleic acid is normallyfound. As used herein therefore, by way of example only, a recombinantcell line constructed with a polynucleotide of the invention makes useof the “isolated” nucleic acid. Alternatively if a particular protein ora specific immunogenic fragment is claimed or used as a vaccine it wouldbe considered to be isolated because it had been identified, separatedand to some extent purified as compared to how it may exist in nature.If the protein or a specific immunogenic fragment thereof is produced ina recombinant bacterium or eukaryote expression vector that produces theantigen it is considered to exist as an isolated protein or nucleicacid. Example, a recombinant cell line constructed with a polynucleotidemakes use of an “isolated” nucleic acid.

“Metabolizable adjuvant” Adjuvants consisting of components that arecapable of being metabolized by the target species such as vegetable oilbased adjuvants. A Metabolizable adjuvant may be a metabolizable oil.Metabolizable oils are fats and oils that typically occur in plants andanimals and usually consist largely of mixtures of triacylglycerols,also known as triglycerides or neutral fats. These nonpolar, waterinsoluble substances are fatty acid triesters of glycerol.Triacylglycerols differ according to the identity and placement of theirthree fatty acid residues. Compare to “Non-metabolizable adjuvant”

“Non-metabolizable adjuvant” Adjuvants consisting of components thatcannot be metabolized by the body of the animal subject to which theemulsion is administered. Non-metabolizable oils suitable for use in theemulsions of the present invention include alkanes, alkenes, alkynes,and their corresponding acids and alcohols, the ethers and estersthereof, and mixtures thereof. Preferably, the individual compounds ofthe oil are light hydrocarbon compounds, i.e., such components have 6 to30 carbon atoms. The oil can be synthetically prepared or purified frompetroleum products. Preferred non-metabolizable oils for use in theemulsions of the present invention include mineral oil, paraffin oil,and cycloparaffins, for example. The term “mineral oil” refers to anon-metabolizable adjuvant oil that is a mixture of liquid hydrocarbonsobtained from petrolatum via a distillation technique. The term issynonymous with “liquefied paraffin”, “liquid petrolatum” and “whitemineral oil.” The term is also intended to include “light mineral oil,”i.e., oil which is similarly obtained by distillation of petrolatum, butwhich has a slightly lower specific gravity than white mineral oil. See,e.g., Remington's Pharmaceutical Sciences, 18^(th) Edition (Easton, Pa.:Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil canbe obtained from various commercial sources, for example, J. T. Baker(Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferredmineral oil is light mineral oil commercially available under the nameDRAKEOL®.

“Monoclonal antibody” refers to antibodies produced by a single line ofhybridoma cells, all directed towards one epitope on a particularantigen. The antigen used to make the monoclonal antibody can beprovided as an isolated protein of the pathogen or the whole pathogen. A“hybridoma” is a clonal cell line that consists of hybrid cells formedby the fusion of a myeloma cell and a specific antibody-producing cell.In general, monoclonal antibodies are of mouse origin; however,monoclonal antibody also refers to a clonal population of an antibodymade against a particular epitope of an antigen produced by phagedisplay technology or method that is equivalent to phage display orhybrid cells of non-mouse origin.

“N days” or “M-days” following an event refers, respectively, to anytime on the Nth or Mth day after the event. For example, vaccinating asubject with a second vaccine 14 days following administration of afirst vaccine means that the second vaccine is administered at any timeon the 14th day after the first vaccine.

“ORF” indicates “open reading frame”, i.e. the coding region of a gene.

“Oral” or “peroral” administration refers to the introduction of asubstance, such as a vaccine, into a subject's body through or by way ofthe mouth and involves swallowing or transport through the oral mucosa(e.g., sublingual or buccal absorption) or both. Intratracheal is alsoan oral or peroral administration.

“Oronasal” administration refers to the introduction of a substance,such as a vaccine, into a subject's body through or by way of the noseand the mouth, as would occur, for example, by placing one or moredroplets in the nose. Oronasal administration involves transportprocesses associated with oral and intranasal administration.

“Parenteral administration” refers to the introduction of a substance,such as a vaccine, into a subject's body through or by way of a routethat does not include the digestive tract. Parenteral administrationincludes subcutaneous administration, intramuscular administration,transcutaneous administration, intradermal administration,intraperitoneal administration, intraocular administration, andintravenous administration. For the purposes of this disclosure,parenteral administration excludes administration routes that primarilyinvolve transport of the substance through mucosal tissue in the mouth,nose, trachea, and lungs.

“Pharmaceutically acceptable” refers to substances, which are within thescope of sound medical judgment, suitable for use in contact with thetissues of subjects without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit-to-riskratio, and effective for their intended use.

“Pharmaceutically acceptable carrier” refers to a carrier medium thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredient and is not toxic to the subject to whom it isadministered.

“Polyclonal antibody” refers to a mixed population of antibodies madeagainst a particular pathogen or antigen. In general, the populationcontains a variety of antibody groups, each group directed towards aparticular epitope of the pathogen or antigen. To make polyclonalantibodies, the whole pathogen or an isolated antigen is introduced byinoculation or infection into a host that induces the host to makeantibodies against the pathogen or antigen.

“Preventing infection” means to prevent or inhibit the replication ofthe bacteria or virus which cause the identified disease, to inhibittransmission of the bacteria or virus, or to prevent the bacteria orvirus from establishing itself in its host, or to alleviate the symptomsof the disease caused by infection. The treatment is consideredtherapeutic if there is a reduction in bacterial or viral load.

“Protection”, “Protecting”, and the like, as used herein with respect toa vaccine, means that the vaccine prevents or reduces the symptoms ofthe disease caused by the organism from which the antigen(s) used in thevaccine is derived. The terms “protection” and “protecting” and thelike, also mean that the vaccine can be used to “treat” the disease orone of more symptoms of the disease that already exists in a subject.

“Respiratory” administration refers to the introduction of a substance,such as a vaccine, into a subject's body through or by way of inhalationof a nebulized (atomized) substance. In respiratory administration, theprimary transport mechanism involves absorption of the atomizedsubstance through the mucosa in the trachea, bronchi, and lungs and istherefore different than intranasal or peroral administration.

“Specific for,” when used to describe antibodies of the invention,indicates that the variable regions of the antibodies of the inventionrecognize and bind a specific H3N8 strain exclusively (i.e., are able todistinguish a particular H3N8 protein from other known proteins byvirtue of measurable differences in binding affinity, despite theexistence of localized sequence identity, homology, or similaritybetween H3N8 proteins and such polypeptides). It will be understood thatspecific antibodies may also interact with other proteins (or otherantibodies in ELISA techniques) through interactions with sequencesoutside the variable region of the antibodies, and, in particular, inthe constant region of the molecule. Screening assays to determinebinding specificity of an antibody of the invention are well known androutinely practiced in the art. For a comprehensive discussion of suchassays, see Harlow et al. (Eds.), Antibodies: A Laboratory Manual; ColdSpring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.Antibodies of the invention can be produced using any method well knownand routinely practiced in the art.

“Subunit vaccine” refers to a type of vaccine that includes one or moreantigens, but not all antigens, which are derived from or homologous to,antigens from a pathogen of interest, such as a virus, bacterium,parasite or fungus. Such a composition is substantially free of intactpathogen cells or pathogenic particles, or the lysate of such cells orparticles. Thus, a subunit vaccine can be prepared from at leastpartially purified, or substantially purified, immunogenic polypeptidesfrom the pathogen or its analogs. Methods of obtaining an antigen orantigens in the subunit vaccine include standard purificationtechniques, recombinant production, or chemical synthesis. A “Subunitvaccine” thus refers to a vaccine consisting of a defined antigeniccomponent or components of a complete viral, bacterial or otherimmunogen.

“Specific immunogenic fragment” is meant a portion of a sequence that isrecognizable by an antibody that is specific for the sequence.

“Subject” refers to any animal having an immune system, which includesmammals such as dogs.

“TCID₅₀₃₈ refers to “tissue culture infective dose” and is defined asthat dilution of a virus required to infect 50% of a given batch ofinoculated cell cultures. Various methods may be used to calculateTCID₅₀, including the Spearman-Karber method which is utilizedthroughout this specification. For a description of the Spearman-Karbermethod, see B. W. Mahy & H. O. Kangro, Virology Methods Manual 25-46(1996).

“Therapeutic agent” refers to any molecule, compound, virus ortreatment, preferably a virus attenuated or killed, or subunit orcompound, that assists in the treatment of a viral infection or adisease or condition caused thereby.

“Therapeutically effective amount,” in the context of this disclosure,refers to an amount of an antigen or vaccine that would induce an immuneresponse in a subject (e.g., dog) receiving the antigen or vaccine whichis adequate to prevent or ameliorate signs or symptoms of disease,including adverse health effects or complications thereof, caused byinfection with a pathogen, such as a virus (e.g., H3N8), bacterium,parasite or fungus. Humoral immunity or cell-mediated immunity or bothhumoral and cell-mediated immunity may be induced. The immunogenicresponse of an animal to a vaccine may be evaluated, e.g., indirectlythrough measurement of antibody titers, lymphocyte proliferation assays,or directly through monitoring signs and symptoms after challenge withwild type strain. The protective immunity conferred by a vaccine can beevaluated by measuring, e.g., reduction in clinical signs such asmortality, morbidity, temperature number and overall physical conditionand overall health and performance of the subject. The amount of avaccine that is therapeutically effective may vary depending on theparticular virus used, or the condition of the subject, and can bedetermined by one skilled in the art.

“Transmitted” means a virus that is capable of being passed from a firstanimal (dog) to a second animal (dog) where the second dog demonstratesserovonversion to the transmitted virus.

“Treating” refers to reversing, alleviating, inhibiting the progress of,or preventing a disorder, condition or disease to which such termapplies, or to preventing one or more symptoms of such disorder,condition or disease.

“Treatment” refers to the act of “treating” as defined immediatelyabove.

“Vaccine” refers to an immunogenic composition selected from a virus,either modified live, attenuated, or killed, or a subunit vaccine, orany combination of the aforementioned. Administration of the vaccine toa subject results in an immune response. The vaccine may be introduceddirectly into the subject by any known route of administration,including parenterally, perorally, and the like.

Part 1. Antigens and Virus Strains, Their Production, Manufacture,Formulation Into and Administration of Vaccines.

One aspect of the present invention provides vaccines that use thefollowing antigens to provoke an immunogenic response.

Useful Antigen(s) of the invention. The antigens of this invention maybe any identified influenza virus strain, from any bird or mammal,including but not limited to, influenza virus having the subtype H3hemagglutinin and subtype N8 neuraminidase, or the H3N8 subtype or morecommonly referred to as an H3N8 virus. The influenza may be of anymammalian or avian origin, including but not limited to swine, equine orcanine origin. Equine and canine influenza antigens are preferred.Strains having the subtype glycoproteins designated H3 or N8 and morepreferably strains having both H3 and N8.

The strains and variants and mutants and variants thereof are alsopreferred as described in Transmission of Equine Influenza to Dogs (P.C. Crawford et al., Science 310, 482-485 (2005). The viral HA is acritical determinant of host species specificity of influenza virus.

The influenza antigens of this invention can be isolated from dogs,horses, pigs, and fowl both domestic and wild. The animals chosen forsample collection should display acute and/or sub-acute clinicalsyndromes which may include mild to severe respiratory symptoms andfever. Animals may also exhibit signs of anorexia and lethargy. Methodsof virus isolation are well known to those skilled in the art including:inoculating mammalian or avian cell cultures, embryonated eggs withnasal or pharyngeal mucus samples from clinical specimens, collection byswabbing of the nasal passage or throat, or by collecting tissues suchas: spleen, lung, tonsil and liver and lung lavage. The cytopathiceffect of the virus can be observed in cell culture, and allantoic fluidor cell lysates can be tested for their ability to agglutinate human,rooster, turkey or guinea pig red blood cells, presumptive evidence forthe presence of an influenza virus.

Nomenclature of viral strains and possible antigens. Type A influenzavirus strains are subdivided into subtypes based on the antigeniccharacteristics of their glycoproteins on the virion surface. Thesevirus glycoproteins are hemagglutinin (HA) and neuraminidase (NA).Typically the HA subtype is named first and the NA second, thus, H3N8refers to a virus with hemagglutinin subtype 3 and neuraminidase subtype8. The subtype is based on serological analysis of the HA and NA. Usingthe procedures disclosed herein a vaccine for any of these subtypes maybe made. Currently there are 16 identified HA subtypes and 9 identifiedNA subtypes. There may be more in the wild that have not yet beendescribed. Specifically, identified subtypes include H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 and N1, N2, N3, N4,N5, N6, N7, N8, and N9. All of these combinations of subtypes, and anycombination thereof and any subtype and combination of future subtypesthat will be identified in the future using the procedures describedabove or substantially similar procedures are hereby described andclaimed as useful antigens of this invention. All other HA and NAcombinations of subtypes are disclosed. This includes but is not limitedto preferred subtypes H3N8 and H7N7.

The influenza virus hemagglutinin (HA) is the virion surfaceglycoprotein that attaches the virus to its receptors on host cells andfuses the viral envelope with the membranes of endocytic vesicles toinitiate the infectious process. It is also the virion component mostimportant in the stimulation and formation of protective antibodies. Theamino acid sequence of the HA and hence the location of itsN-glycosylation sites is determined by the viral genome.

The segmented, negative stranded RNA genome of the influenza virus isreplicated by an RNA dependent-RNA polymerase which lacks an effectiveproofreading function, leading to a high rate of transcription errorsthat can result in amino acid substitutions in surface glycoproteins HAand NA. One of the consequences of this high mutation frequency is thatvirus populations contain mutants that differ from the majority in thenumber and position of the N-linked glycans on the HA. The structures ofthese oligosaccharides may be determined by their position on the HA andby the array of biosynthetic and trimming enzymes provided by the hostcell in which the virus is grown. Thus, the plasticity of the viralgenome and the host-specified glycosylation machinery can, together,create virus populations that are more heterogeneous in structure andfunction than could be developed by either process alone. This diversityis considered to be responsible for survival of these viruses in avariety of biological niches and for their ability to overcome theinhibitory effects of neutralizing antibodies and antiviral agents.Mutations in the viral genome of various strains have been identifiedand those mutated strains are also claimed here. For example, some ofthese mutants are described in Transmission of Equine Influenza to Dogs(P. C. Crawford et al., Science 310, 482-485 (2005) incorporated hereinby reference.

This invention also discloses a vaccine made from a specific straincollected and identified as Equine Influenza StrainA/Equine/2/Miami/1/63. This strain is deposited at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209as ATCC accession number VR 317. This strain was originally isolatedfrom nasal washings from a sick horse in Miami in 1963. The virus waspassaged 5 times in chicken embryos. The virus is further classified asH3N8.

Another example of a North American H3N8 influenza virus derived from ahorse is A/Equine/Kentucky/1998. Additional examples of H3N8 derivedfrom a horse are A/Equine/Kentucky/15/2002, A/Equine/Ohio/1/2003,A/Equine/Kentucky/1/1994, A/Equine/Massachusetts/213/2003,A/Equine/Wisconsin/2003 and A/Equine/New York/1999. Other examples areEuropean H3N8 influenza virus derived A/Equine/Newmarket/A2/1993.

This invention also discloses a vaccine made from a specific straincollected and identified as canine Influenza StrainA/canine/Iowa/13628/2005 and Strain A/canine/Iowa/9A1/B5/08/D12. Thelatter strain, Strain A/canine/Iowa/9A1/B5/08/D12 was deposited as UC25508, on 29 Jun. 2006 at the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, the ATCC accessionnumber is PTA-7694. The virus is further classified as H3N8.

In addition to the above strains we disclose a strain obtained in thefollowing manner. Identify a dog or group of dogs exhibiting clinicalsigns of respiratory disease, obtain samples of oral or nasal secretionsor samples derived from respiratory tissue or internal organ tissue fromthe dogs, assay the samples and identify the presence of an H3N8influenza virus. Using the procedures described herein, isolate, purify,culture, grow, produce, concentrate this virus antigen and identify asPfizer canine influenza virus. Adapt to and passage in eitherembryonated eggs or canine cells or both, identify as Master Seed,canine influenza H3N8 virus. A canine derived H3N8 influenza virus ispreferred. An equine or porcine derived H3N8 can also be used as well asan influenza virus of subtype H3 or N8. We disclose a strain obtained inthe following manner. Infect a dog or group of dogs with equineinfluenza H3N8. From dogs exhibiting clinical or subclinical signs ofrespiratory disease, obtain samples of oral or nasal secretions, orsamples derived from respiratory tissue or lung lavage or internal organtissue from the dogs, assay the samples and identify the presence of anH3N8 influenza virus. Using the procedures described herein, isolate,purify, culture, grow, produce, concentrate this virus antigen andidentify as canine influenza virus. Adapt to and passage in eitherembryonated eggs or canine cells or both, identify as Master Seed,canine influenza H3N8 virus. A canine derived H3N8 influenza virus ispreferred. An equine or porcine derived H3N8 can also be used as well asan influenza virus of subtype H3 or N8. The porcine derived H3N8 virusis treated in the same manner as the equine or canine derived virus thatis fully described herein.

Part 2. Detailed Descriptions of the Production, Manufacture,Formulation and Administration of Vaccines Produced From the Antigens ofPart 1.

Part 2a) Discussion. The virus antigens of Part 1, may be made intouseful compositions of matter comprising the virus antigen modified toreduce its virulence and formulated into a useful formulation or vaccineformulation. The following descriptions provide details for theproduction, manufacture, formulation and administration of vaccinesuseful for the prevention or treatment of clinical signs associated withinfluenza virus infection in dogs, or in the prevention of disease indogs caused by an canine or equine influenza virus. The canine influenzainfection to be treated may be caused by equine influenza virus or itmay be a new modified canine influenza derived from a equine influenzavirus. The treatments described here may act as an aid in the preventionof shedding of canine or equine influenza virus in canine disease.

Described herein are methods and materials for treating and immunizinganimals with a vaccine and in particular dog against equine and canineinfluenza viruses. The method includes administering to the dog atherapeutically effective amount of a first, second and or third vaccinethat is capable of inducing an immune response, and in particular in thedog against H3N8 influenza viruses. The vaccine of the present inventionis generally intended to be a prophylactic treatment which immunizesdogs against disease caused by virulent strains of equine or canineinfluenza virus.

Here we disclose vaccines that provide active and or passive immunity.Either the entire vaccine, or specific immunogenic fragments of theirproteins, would be expected to be effective when given as a therapeutictreatment against equine or canine influenza viruses. Thus, the immunitythat is provided by the present invention can be either active orpassive immunity, and the intended use of the vaccine can be eitherprophylactic or therapeutic. In a preferred embodiment, the vaccinefurther includes a vaccine for immunizing a dog against any form of acanine or equine influenza virus.

Part 2 b) Vaccine Production and Antigen Concentration. The vaccinedescribed in this section may be produced by growing the selected virusin cells. Production of the virus is preferred in equine or caninemammalian cell culture. Virus (antigen) growth or production in eggs isalso preferred. Dog kidney cell lines are preferred. Viral propagationmay also be accomplished on any useful media and permissive cell lines,which may be derived from avian or mammalian cell lines derived fromfeline, equine, bovine or porcine cell lines. The vaccines typicallycontain between 10³ and 10⁹ TCID₅₀, levels of virus prior toinactivation. Alternatively the antigen content in the virus preparationcould be assayed by the hemagglutination inhibition (HI) test, singleradial diffusion or hemagglutination assay and using this assay onewould prefer a vaccine with a titer of between 10 to 10,000 HA units/ml,more typically between 100 to 2000 HA units/ml, and frequently between100 to 1000 HA units/ml as the amount administered per dose.

Virus growth: cell lines and embryonated eggs The preferred cell linefor propagation of influenza virus is canine kidney (DK). Other celllines can be utilized which include primary and immortalized equinekidney (EK), equine dermal (ED), swine testicular (ST), porcine kidney(PK), bovine kidney (BK), feline kidney (FK), Vero and primary andimmortalized chicken embryo fibroblasts (CEF). The preferred cellculture system for growing influenza virus is a traditional adherentmonolayer culture. Alternatively, suspension and microcarrier cellculture systems can also be utilized. A preferred microcarrier isCytodex 3 microcarrier beads (Amersham Biosciences Ltd.). Other examplesof microcarriers include beads composed of glass, silicone and dextran,DEAE, collagen, dextran or gelatin.

The preferred vessel for culturing cell lines and propagation ofinfluenza virus is the roller bottle, the preferred roller bottlesurface area is 1760 cm² but can range from 490-4250 cm². Alternatively,other useful cell culture formats include flasks (150 cm²-420 cm²),stacked modules (21,000 cm²-340,000 cm²) and stir tanks (1.0 L-900 L).The preferred multiplicity of infection (MOI) is 0.001-0.1 but can rangefrom 0.0001-2.0. The preferred window to harvest virus from cell cultureis day 2 to 5 post-infection, but can range from day 1 to day 7post-infection.

Virus propagation can also be accomplished by inoculating embryonatedeggs. Typically 0-12 day old embryonated eggs are used for viruspropagation. Preferrably 7-8 day old embryonated eggs are used for virusgrowth. The virus is inoculated into the amniotic cavity of the egg. Thevirus replicates in the cells of the amniotic membrane and largequantities are released back into the amniotic fluid. After 2-3 dayspost inoculation, virus in the amniotic fluid can be harvested.

Cell culture media: Preferred cell culture media formulations topropagate influenza virus includes, but is not limited to, thefollowing: Dulbecco's modified eagle media (DMEM), basal modified eaglemedia, Optimem and Leibovitz-15 (L-15) media. Typically the cell culturemedia is supplemented with 0.1 to 10 units of trypsin. Alternatively,plant derived equivalents of trypsin (e.g. Accutase) ranging from 2-100units can also be used in cell culture for efficient propagation ofvirus. Cell culture media can be used in the absence or presence ofanimal-derived components. An example of supplementation with ananimal-derived component is gamma-irradiated serum ranging from 0.5-10%final concentration.

Part 2c) Vaccine Preparation Inactivated or Killed, Subunit andAttenuated, Modified-Live.

Inactivated or Killed. In one embodiment of the present invention, thevaccine comprises an inactivated or killed H3N8 influenza virus vaccinecomprising an H3N8 equine or canine strain selected from any equine orcanine infectious influenza strain. See Transmission of Equine Influenzato Dogs (P. C. Crawford et al., Science 310, 482-485 (2005). The vaccinecan also be comprised of influenza H3N8 derived from swine or anyinfluenza of subtype H3 or N8. The inactivated vaccine is made bymethods well known in the art. For example, once the virus is propagatedto high titers, it would be readily apparent to those skilled in the artthat the virus antigenic mass could be obtained by methods well known inthe art. For example, the virus antigenic mass may be obtained bydilution, concentration, or extraction. All of these methods have beenemployed to obtain appropriate viral antigenic mass to produce vaccines.The virus may be inactivated by treatment with formalin (e.g. 0.1-10%),betapropriolactone (BPL) (e.g. 0.01-10%), or with binary ethyleneimine(BEI) (e.g. 1-10 mM) which is preferred here, or using other methodsknown to those skilled in the art. Commonly used conditions and agentsare suggested but other agents and concentrations should be apparent toone skilled in the art.

In addition to killed virus production detailed above, various means ofattenuation are also possible and are well known and described in theart and applicable here. Attenuation leading to modified live vaccinesis also possible. Some of these techniques are described here and below.Among the more preferred forms of attenuation are continuous passagingin cell culture, continuous passaging in animals, various methods forgenerating genetic modifications and ultraviolet or chemicalmutagenesis.

Subunit Vaccines. In addition, equine or canine influenza subunitvaccines can be produced by recombinant expression techniques whichinclude, but are not limited to, heterologous prokaryotic expression(e.g., E. coli, Pseudomonas, Salmonella etc.) and heterologouseukaryotic expression (e.g., yeast [Pichia, Yarrowia], insect cells[Baculovirus], etc.) and viral vectors (e.g., canine adenovirus, humanadenovirus, poxvirus, canine herpesvirus).

Attenuated and Modified-Live. An Attenuated Virus Canine Vaccine isprepared from cell line or egg cultivated influenza virus preferably aninfluenza derived H3N8, that has been attenuated by serial passageincluding serial passage at sub-optimal temperatures to a state where itis no longer capable of causing disease, but still capable of elicitinga protective immune response.

Attenuation of an influenza virus may be achieved by serial passaging ofa wild-type influenza virus strain in cell culture. The virus strain canbe passaged in a variety of cell systems until its ability to producedisease is lost whilst its immunogenic character is fully retained. Onceinoculated into the host, the virus may be capable of multiplication tosome extent. Suitable attenuated viral strains may also be obtained byserial passaging to obtain an over-attenuated strain. The“over-attenuation” means that the number of passages for attenuation hasbeen substantially greater than what is normally necessary for theremoval of pathogenicity. The attenuated virus retains its antigenicityafter these numerous passages, for example retaining both itshaemagglutinin and neuraminidase antigens, so that its immunogenicability is not impaired. Such strains produce practically no symptoms orside effects when administered, and thus are safe and efficaciousvaccines.

Attenuation of influenza virus may be achieved through cold-adaptationof an influenza virus strain. Cold-adapted influenza virus strains maybe produced by methods which includes passaging a wild-type influenzavirus, followed by selection for virus that grows at a reducedtemperature. Cold-adapted influenza viruses can be produced, forexample, by sequentially passaging a wild-type influenza virus inembryonated chicken eggs at progressively lower temperatures, therebyselecting for certain members of the virus mixture which stablyreplicate at the reduced temperature. A cold-adapted influenza virusstrain may exhibit a temperature sensitive phenotype. A temperaturesensitive cold-adapted influenza virus replicates at reducedtemperatures, but no longer replicates or forms plaques in tissueculture cells at certain higher growth temperatures at which thewild-type virus will replicate and form plaques. A temperature at whicha temperature sensitive virus will grow is referred to herein as a“permissive” temperature for that temperature sensitive virus, and ahigher temperature at which the temperature sensitive virus will notgrow, but at which a corresponding wild-type virus will grow, isreferred to herein as a “non-permissive” temperature for thattemperature sensitive virus. For example, certain temperature sensitivecold-adapted influenza viruses replicate in embryonated chicken eggs ata temperature at or below about 30° C., and will form plaques in tissueculture cells at a permissive temperature of about 34° C., but will notform plaques in tissue culture cells at a non-permissive temperature ofabout 37° C. Certain cold-adapted influenza viruses may have a dominantinterference phenotype. That is, they dominate an infection whenco-infected into cells with another influenza virus, thereby impairingthe growth of that other virus. A cold-adapted influenza virus may alsobe produced through recombinant means. In this approach, one or morespecific mutations, associated with identified cold-adaptation,attenuation, temperature sensitivity, or dominant interferencephenotypes, are identified and are introduced back into a wild-typeinfluenza virus strain using a reverse genetics approach. Reversegenetics entails using RNA polymerase complexes isolated from influenzavirus-infected cells to transcribe artificial influenza virus genomesegments containing the mutation(s), incorporating the synthesized RNAsegment(s) into virus particles using a helper virus, and then selectingfor viruses containing the desired changes.

Part 2 d) Vaccine Adjuvants, Formulations, Forms and Carriers.Components of vaccines presented here will preferably include one ormore adjuvants. Adjuvants include, but are not limited to, the RIBIadjuvant system (Ribi Inc.) Aluminum salts, including Alum (0.5-20%,more preferred is less than 10%, more preferred are 2 and 5%), Aluminumphosphate (0.5-20%, more preferred is less than 10%, more preferred are2 and 5%), Aluminum hydroxide (Alhydrogel or Rehydragel ranging from0.5-20%, more preferred is less than 10%, more preferred are 2 and 5%),cholesterol, oil-in water emulsions, water-in-oil emulsions such as,e.g., Freund's complete and incomplete adjuvants, Block co-polymer(CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN®adjuvant, saponin, and saponins such as, Quil A, QS-21 (CambridgeBiotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals,Inc., Birmingham, Ala.) with preferred Saponin concentrations of 10-100microgram and about 50 microgram preferred or other saponin fractions,monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labileenterotoxin from E. coli (recombinant or otherwise), cholera toxin, ormuramyl dipeptide, among many others. The immunogenic compositions canfurther include one or more other immunomodulatory agents such as, e.g.,interleukins, interferons, or other cytokines. The immunogeniccompositions can also include gentamicin and Merthiolate.

Components of vaccines may include pharmaceutically acceptableexcipients, including carriers, solvents, and diluents, isotonic agents,buffering agents, stabilizers, preservatives, immunomodulatory agents(e.g., interleukins, interferons, and other cytokines),vaso-constrictive agents, antibacterial agents, antifungal agents, andthe like. Typical carriers, solvents, and diluents include water,saline, dextrose, ethanol, glycerol, and the like. Representativeisotonic agents include sodium chloride, dextrose, mannitol, sorbitol,lactose, and the like. Useful stabilizers include gelatin, albumin, andthe like.

H3N8 influenza virus vaccines are provided in various forms, dependingon the route of administration, storage requirements, and the like. Forexample, the vaccines can be prepared as aqueous solutions ordispersions suitable for use in syringes, droppers, nebulizers, etc., orcan be prepared as lyophilized powders, which are reconstituted insaline, HEPES buffer, or the aqueous, immunogenic fraction of a secondcanine vaccine and the like, prior to use.

The vaccine for any one of the embodiments of the present invention isformulated in a pharmaceutically accepted carrier according to the modeof administration to be used. One skilled in the art can readilyformulate a vaccine that comprises a live or killed equine or canineinfluenza or an immunogenic fragment thereof, a recombinant virus orbacterial vector encoding equine or canine influenza, a specificimmunogenic fragment thereof, or a DNA molecule encoding equine orcanine influenza or a specific immunogenic fragment thereof.

In cases where intramuscular injection is preferred, an isotonicformulation is preferred. Generally, additives for isotonicity caninclude sodium chloride, dextrose, mannitol, sorbitol, and lactose. Inparticular cases, isotonic solutions such as phosphate buffered salineare preferred. The formulations can further provide stabilizers such asgelatin and albumin. In some embodiments, a vaso-constrictive agent isadded to the formulation. The pharmaceutical preparations according tothe present invention are provided sterile and pyrogen-free. However, itis well known by those skilled in the art that the preferredformulations for the pharmaceutically accepted carrier which comprisethe vaccines of the present invention are those pharmaceutical carriersapproved in the regulations promulgated by the United States Departmentof Agriculture, or equivalent government agency in a foreign countrysuch as Canada or Mexico or any one of the European nations for anycanine vaccine, polypeptide (antigen) subunit vaccines, recombinantvirus vector vaccines, and DNA vaccines. Therefore, the pharmaceuticallyaccepted carrier for commercial production of the vaccine of the presentinvention is a carrier that is already approved or will be approved bythe appropriate government agency in the United States of America orforeign country. The vaccine can further be mixed with an adjuvant thatis pharmaceutically acceptable. In certain formulations of the vaccineof the present invention, the vaccine is combined with other caninevaccines to produce a polyvalent vaccine product that can protect canineagainst a wide variety of diseases caused by other canine pathogens.

The vaccine compositions optionally may include vaccine-compatiblepharmaceutically acceptable (i.e., sterile and non-toxic) liquid,semisolid, or solid diluents that serve as pharmaceutical vehicles,excipients, or media. Diluents can include water, saline, dextrose,ethanol, glycerol, and the like. Isotonic agents can include sodiumchloride, dextrose, mannitol, sorbitol, and lactose, among others.Stabilizers include albumin, among others. Any adjuvant known in the artmay be used in the vaccine composition, including metabolizable andnon-metabolozable adjuvants, oil-based adjuvants such as Freund'sComplete Adjuvant and Freund's Incomplete Adjuvant, mycolate-basedadjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide(LPS), peptidoglycans (i.e., mureins, mucopeptides, or glycoproteinssuch as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans(e.g., extracted from Klebsiella pneumoniae), streptococcal preparations(e.g., OK432), Biostim™ (e.g., 01K2), the “Iscoms” of EP 109 942, EP 180564 and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutraloils (such as miglyol), vegetable oils (such as arachis oil), liposomes,and Pluronic® polyols.

The immunogenic compositions of the present invention can be made invarious forms depending upon the route of administration. For example,the immunogenic compositions can be made in the form of sterile aqueoussolutions or dispersions suitable for injectable use, or made inlyophilized forms using freeze-drying techniques. Lyophilizedimmunogenic compositions are typically maintained at about 4° C., andcan be reconstituted in a stabilizing solution, e.g., saline or/andHEPES, with or without adjuvant.

In addition, the immunogenic and vaccine compositions of the presentinvention can include one or more pharmaceutically-acceptable carriers.As used herein, “a pharmaceutically-acceptable carrier” includes any andall solvents, dispersion media, coatings, adjuvants, stabilizing agents,diluents, preservatives, antibacterial and antifungal agents, isotonicagents, adsorption delaying agents, and the like. The carrier(s) must be“acceptable” in the sense of being compatible with the components of theinvention and not deleterious to the subject to be immunized. Typically,the carriers will be will be sterile and pyrogen-free.

Part 2 e) Vaccine Dosages and Assays. Dose sizes of H3N8 influenza virusvaccines typically range in volume from about 2.0 to 0.1 ml depending onthe route of administration. The inactivated vaccines typically containbetween 103 and 109 TCID₅₀, levels of virus prior to inactivation.Alternatively the antigen content in the virus preparation would prefera vaccine with a titer of between 10 to 10,000 HA units/ml, it may have100 to 2000 HA units/ml, and more preferably has between 100-1000 HAunits/ml as the amount administered per dose. For vaccines containingmodified live viruses or attenuated viruses, a therapeutically effectivedose will generally range from about 10⁵ TCID₅₀ to about 10⁸ TCID₅₀,inclusive. For vaccines containing subunit antigens, such as influenzaH3 or N8 proteins, a therapeutically effective dose generally rangesfrom about 10 μg to about 100 μg, inclusive. While the amounts andconcentrations of adjuvants and additives useful in the context of thepresent invention can readily be determined by the skilled artisan, thepresent invention contemplates compositions comprising from about 50 μgto about 2000 μg of adjuvant and preferably about 500 μg/2 ml dose ofthe vaccine composition.

Assays. Influenza virus can be detected by virus isolation or by viralantigen, viral RNA or specific antibody detection methods. Methods usedto detect virus or viral components include immunofluorescence of lungtissue, nasal epithelial cells or bronchioalveolar lavage contents,immunohistochemistry of tissue samples, enzyme linked immunosorbentassay (ELISA), polymerase chain reaction (PCR), cell culture andimmunoperoxidase, fluorescent antibody staining for determination ofvirus type and subtype, and a rapid enzyme-immunoassay membrane test.Tissues typically assessed for influenza virus include lungs, lunglavage, tonsils, trachea, spleen as well as serum. Monoclonal antibodiesare also available that specifically target various virus epitopes,namely haemagglutinin (HA) and neuraminidase (NA) epitopes. The mostcommon serologic assay for diagnosis of influenza is the haemagglutinininhibition (HI) assay. One of its advantages is that it can discriminatebetween different subtypes and antigenic variants within a subtype. TheHI assay can be performed using equine, canine, swine or avian derivedsera. A more precise method for measuring antibody is by single radialhemolysis (SRH) technique. SRH is more sensitive than HI assays and hasa greater degree of precision. A 50% increase in zone area represents arise in antibody and is evidence of recent infection.

Part 2 f) Timing and Routes of Administration. Inoculation of a dog ispreferably made to a dog that is 6 weeks and more preferably 8 weeks orolder. The dogs should receive preferably 2 dosages, each typicallyadministered 3-4 weeks apart, preferably 3 weeks, via subcutaneousinjection (SC) depending on the condition of the dog and itsenvironment. This would be to obtain a full, broad immunogenic response.In another embodiment of the present invention, the dog is subjected toa series of 3 vaccinations to produce a full, broad immune response.Annual revaccination with a single dose is recommended. The dogs mayoptionally be given a booster at 3 and or 6 months if needed. Thepreferred route is subcutaneous injection, using about 1 mL, butintramuscular (IM), using about 1 mL, or intradermal (ID), using 0.1-0.3mL, oral, oronasal or nasal routes, using 0.2 to 0.5 mL, are alsopreferred. Other times, injection sites, amounts used and type ofadministration will be apparent to one of ordinary skill in the art.

The route of administration for any one of the embodiments of thevaccine of the present invention includes, but is not limited to,intradermal, intramuscular, intraocular, intraperitoneal, intravenous,oral, oronasal, and subcutaneous, as well as inhalation, suppository, ortransdermal. The preferred routes of administration include intradermal,intramuscular, intraperitoneal, oronasal, and subcutaneous injection.The vaccine can be administered by any means that includes, but is notlimited to, syringes, nebulizers, misters, needleless injection devices,or microprojectile bombardment gene guns (Biolistic bombardment).

Part 3. Specific Descriptions of the Production, Manufacture,Formulation and Administration of Selected Vaccines.

Here we provide more detailed specific descriptions of a killed orinactivated monovalent vaccine, for the treatment of canine influenza.

Part 3a) Killed or Inactivated Virus Monovalent Canine Influenza VaccineUsing any of the antigens described in Part 1 above, including preferredinfluenza derived H3N8 antigen derived from Canine or Equine Influenza,including inactivated and adjuvanted cultures of H3N8 influenza virus.

Killed or Inactivated Virus Bi-Valent Canine Influenza Vaccine

Using any of the antigens described in Part 1 above, preferably aninfluenza derived H3N8 antigen derived from Canine and Equine Influenza,inactived and adjuvanted cultures of H3N8 influenza virus. Whereas thecanine antigen is derived from one canine influenza virus and the equineantigen is derived from one equine influenza virus. The canine antigencan also consist of two canine influenza viruses or two equine influenzaviruses.

Killed or Inactivated Virus Tri-Valent Canine Vaccine

Using any of the antigens described in Part 1 above, but more preferablyan influenza derived H3N8 antigen derived from Canine or EquineInfluenza, inactived and adjuvanted cultures of H3N8 influenza virus.The following trivalent vaccines are described: a) A canine antigen isderived from one canine influenza virus and an equine antigen is derivedfrom two equine influenza viruses, b) a canine antigen is derived fromtwo canine influenza viruses and an equine antigen is derived from oneequine influenza virus, c) a canine antigen is derived from three canineinfluenza viruses and or an equine antigen is derived from three equineinfluenza viruses. Any combination of the above is described in order tomake a tri-valent vaccine.

Part 3 b) Killed or Inactivated Vaccine Production and AntigenConcentration. The vaccine described in this section may be produced bygrowing the selected virus in cells. Production of the virus ispreferred in equine or canine mammalian cell culture also preferred isvirus (antigen) growth or production in eggs is also preferred. Dogkidney cell lines are more preferred. Viral propagation may also beaccomplished on any useful media, including permissive cell lines, whichmay be derived from feline, equine, bovine, avian or porcine cell lines.The vaccines should contain between 10⁵ and 10⁸ TCID₅₀, levels of virusprior to inactivation. Alternatively the virus preparation could beassayed by the Hemagglutination Inhibition (HI) test or hemagglutinationassay. Using this assay, one would prefer a vaccine with a titer ofbetween 10 to 10,000 HA units/mL, more typically between 100 to 2000 HAunits/mL, and often between 100 to 1000 HA units/mL, as the amountadministered.

Killed or Inactivated Virus growth: cell lines and embryonated eggs Thepreferred cell culture system for growing influenza virus is thetraditional adherent monolayer culture. Alternatively, suspension andmicrocarrier cell culture systems can also be utilized. A preferredmicrocarrier is Cytodex 3 microcarrier beads (Amersham BiosciencesLtd.). The preferred vessel for culturing cell lines and propagation ofinfluenza virus is the roller bottle format, the preferred roller bottlesurface area is 1760 cm² but can range from 850-4250 cm². The preferredmultiplicity of infection (MOI) is 0.001-0.1 but can range from0.0001-2.0. The preferred harvest of virus from cell culture is day 2 to5 post-infection but can range from day 1 to day 7 post-infection.

Cell culture media: Preferred cell culture media formulations topropagate influenza virus growth includes but are not limited to thefollowing: Dulbecco's modified eagle media (DMEM), basal modified eaglemedia, Optimem and Leibovitz-15 (L-15) media. Typically the cell culturemedia is supplemented with 0.1 to 10 units of trypsin.

Part 3c) Killed or Inactivated Inactivation. After production the virusmay be killed or inactivated with any method commonly used in the art. Amore preferred method would be to inactivate the virus fluids with BEI,described below. Also useful is to inactivate the virus containingfluids with formalin or BPL.

Part 3 d) Killed or Inactivated Vaccine Adjuvants, Formulations, Formsand Carriers. The antigens of Part 1 and in particular the inactivatedor killed viruses may be formulated in a variety of ways to produce auseful vaccine. A preferred formulation is to combine killed vaccinewith an adjuvant. Numerous adjuvants may be used with the vaccines ofthis invention, several preferred are noted. The amount of adjuvantstypically comprises from about 25 μg to about 1000 μg, inclusive, of a 1mL dose. Especially useful adjuvants for the Canine Influenza Vaccineare the following, which may also be used in combinations, preferredcombinations noted: Aluminum salts, including Alum (0.5-20%, morepreferred is less than 10%, more preferred are 2 and 5%), Aluminumphosphate (0.5-20%, more preferred is less than 10%, more preferred are2 and 5%), Aluminum hydroxide (Alhydrogel or Rehydragel ranging from0.5-20%, more preferred is less than 10%, more preferred are 2 and 5%),AMPHIGEN® adjuvant, saponins (preferred are Quil A or QS-21 or QA-21ranging from 1-100 ug Cambridge Biotech Inc., Cambridge Mass.), GPI-0100(Galenica Pharmaceuticals, Inc., Birmingham, Ala.) with preferredsaponin concentrations of 10-100 microgram and about 50 microgrampreferred, Cholesterol, or other synthetic polymers, DEAE dextran,Squalene, etc.

Preferred adjuvants and combinations are: 2, 3, 4 or 5% Alum, anycombination of Alum with QuilA, Cholesterol, use of any known commercialvaccine adjuvant and formulation. Especially preferred are 2-5% Alumalone, Quil A alone and Quil A and Cholesterol which is known as “QAC.”QAC may be used alone or in combination with additional Quil A andCholesterol. Other known adjuvants may also be used. The othercomponents of the vaccines may be adjusted to modify the physical andchemical properties of the vaccines. For example, adjuvants typicallycomprise from about 25 μg to about 1000 μg, inclusive, of a 1 mL dose.Similarly, the vaccine described herein could be combined withantibiotics, which may comprise from about 1 μg to about 60 μg,inclusive, of a 1 mL dose. Other ingredients are possible.

Part 3 e) Killed or Inactivated Vaccine Dosages and Assays. Dose sizesof the H3N8 influenza virus vaccines typically range from about 2.0 to0.1 ml, depending on the route of administration. For vaccinescontaining modified live viruses or attenuated viruses, atherapeutically effective dose will generally range from about 10⁵TCID₅₀ to about 10⁸ TCID₅₀, inclusive. For vaccines containing subunitantigens, such as influenza H3 or N8 proteins, a therapeuticallyeffective dose generally ranges from about 10 μg to about 100 μg,inclusive. For vaccines containing inactivated influenza virus thevaccines should contain between 10³ and 10⁹ TCID₅₀, levels of virusprior to inactivation. The vaccine will preferably have between 100-1000HA units/ml as the amount administered per dose. The most preferred isabout 640 HA per dose.

Part 3 f) Killed or Inactivated, Timing and Route of Administration. SeePart 2 f for the timing of the vaccination. The preferred route issubcutaneous injection, SC, using about 1 mL, but intramuscular, 1M, ofabout 1 mL or intradermal using 1.0-0.2 mL and oronasal, and nasal using0.2 to 0.5 mL are also preferred

Part 4. Combination of Vaccines

The antigens and vaccines of this invention can be combined with otherproducts or vaccines. For example, they can be combined in either theliquid or desiccated fraction with Pfizer's Canine Vanguard® line, whichincludes many products, and or Pfizer's Canine Vanguard Plus® line,which also includes many products, including Vanguard Plus CCV/L4® andother combinations to offer complete protection against major puppydiseases. The equine or canine influenza virus antigen can be combinedin any various combinations with the following canine antigens; canineparainfluenza (CPIV), canine distemper virus (CDV), canine parvovirus(CPV), canine adenovirus-1 (CAV-1), canine adenovirus-2 (CAV-2),Leptospira canicola, Leptospira grippotyphosa, Leptospiraicterohaemorrhagiae, Leptospira pomona, Leptospira bratislava, caninerespiratory coronavirus (CRCV), enteric canine coronavirus (CCV), bovinecoronavirus (BCV) and Bordetella bronchiseptica antigen.

In addition, the equine or canine influenza virus antigens and vaccinesof this invention can be combined with Bordetella bronchiseptica (p68 orBronchicine® or Canvac CCi®) and/or canine parainfluenza (CPIV) and/orcanine respiratory coronavirus (CRCV) and/or bovine coronavirus (BCV)vaccines to provide a pre-boarding vaccine to protect against the commonagents of kennel cough. Initial dosage of the combination vaccines wouldbe 2 1 mL doses 3 weeks a part, a single 1 mL booster could be givenprior to subsequent boardings.

Part 5. Specific Examples of Vaccines

The following vaccines are specifically provided.

EXAMPLES 1-3 Mammalian Cell Grown

Cell derived Pfizer Equine H3N8 (A/Equine/2/Miami/1/1963) in a) 2 or 5%Alum or b) in QAC

EXAMPLES 4-6 Mammalian Cell Grown

Cell derived Pfizer clinical isolate Canine H3N8A/Canine/Iowa/9A1/B5/D8/D12, in a) 2 or 5% Alum or b) in QAC.

EXAMPLES 7-9 Avian Egg Grown

Egg derived Pfizer Equine H3N8 in a) 2 or 5% Alum or b) in QAC

EXAMPLES 10-11 Avian Egg Grown

Egg derived Pfizer clinical isolate Canine H3N8 in a) 2 or 5% Alum or b)in QAC

Part 6. Specific Examples of Vaccine Testing Procedures

The vaccines of this invention can be evaluated and confirmed using thefollowing.

Purpose bred research dogs 6 weeks of age or older are administered thevaccines described herein. Dogs are allotted into vaccine groups using 5to 20 dogs per group. Vaccine is administered as two 1 mL doses or two0.5 mL doses 3 weeks apart via the subcutaneous or intramuscular route.Control dogs are given placebo vaccine. Serum samples are collected fromeach dog the day of first and second vaccination then twice weekly postsecond vaccination as well as post challenge. The serum from vaccinateddogs is tested for seroconversion to vaccine antigen via HI(hemagglutinin inhibition) or SRH (single radial hemolysis). Dogs arechallenged with H3N8 virus by aerosol or droplets via the intranasal ororal route (which can include intratracheal) at day 14 or day 28 postsecond vaccination. Dogs are observed post challenge for clinical signsto the disease by monitoring clinical signs such as respiratorysymptoms, fever, anorexia, lethargy. Nasal secretions are collected byswabbing vaccinated dogs and non-vaccinated controls every other daypost challenge for 10-14 days to measure shedding of challenge virus.Presence of shedding virus is confirmed by cell tissue culture(preferably dog kidney). Tissue samples including trachea, tonsil, lungare collected from a subset of dogs post challenge. The presence ofvirus in dog tissue samples is confirmed as described above orimmunohistochemical analysis. By comparing shedding of challenge virusand virus in tissue samples, dogs vaccinated with an effective vaccinewill have a lower virus load or lower virus amount as compared tonon-vaccinated control dogs. An effective vaccine will result in lowervirus load or lower virus amount in a vaccinated animal and vaccinatedanimals will demonstrate a reduction in clinical signs associated withinfluenza infection as compared to an non-vaccinated control.

SPECIFIC EXAMPLES

Challenge and Efficacy Study Evaluation of Canine Influenza Vaccines inDogs. Evaluation of equine Influenza Vaccine, Killed Virus to ProvideCross-Protection in Canines Against Influenza Virus.

To evaluate seroprotection of equine influenza viruses;A/Equine/2/Miami/1/63 and A/Equine/Ohio/1/2003 containing vaccines, 7-10week old dogs were allotted into one of nine treatment groups (Table 1).Dogs were vaccinated with a 1.0 mL dose via the subcutaneous route onStudy Days 0 and 21. Blood for serum was collected from all dogsenrolled in the study on Study Days 0 (first vaccination), 21 (secondvaccination) and 35 (14 days post second vaccination). All serum sampleswere assayed for HAI to Miami63, Ohio03, and CIV. The serologicalresponse data are presented in Table 2. Statistically significantdifferences in serological titers compared to non-vaccinated animalswere demonstrated on Study Day 35 in the following IVP groups: T02, T03,T04, T06, T07, T08, and T09 (Table 2). Additionally, statisticallysignificant differences were observed on Study Day 21 for the followingIVP groups: T06, T07, T08, and T09 (Table 2).

Preliminary Safety

In addition to measuring serological response, preliminary vaccinesafety was evaluated in this study. Over all, no clinically importantvaccine associated reactions were observed with any of the IVPsevaluated. No vaccine associated systemic reactions were observed duringthe course of the study. Additionally, no injection site swellings wereobserved following the first vaccination in any of the treatment groups(Table 3). On Day 21 (prior to 2^(nd) vaccination), small swellings weredetected upon palpation of the injection site in 34 animals from fivetreatment groups. Of these groups, four (T02, T03, T06, and T07) areadjuvanted with 2% Alum, while the fifth treatment group (T09) isadjuvanted with Quil-A/Cholesterol/Plus other adjuvants. The geometricmean volume on this Day 21 swelling ranged in size from 0.07 to 0.24 cm³across treatments.

Following the second vaccination, Study Days 22-24, injection siteswellings were observed in 58 of 87 animals. These swellings peaked 24hours following vaccination (Day 22) and decreased in size towardresolution until Study Day 24. None of the injection site swellings werepainful or hot to touch, while five were found to be hard to tough.

Evaluation of equine Influenza Vaccine, Killed Virus to ProvideCross-Protection in Canines Against Influenza Virus By Challenge

Six of the nine treatment groups were challenged with CIV: T01, T02,T03, T06, T07, and T09. Study Day 0, day of challenge, animals 6 weeksafter the second vaccination with approximately HA of 1:8 per 50 mlrepresenting approximately 6.9 logs per mL of CIV. Dogs were challengedvia the intratracheal route in a model similar to the swine influenzavirus respiratory disease model. Clinical signs associated withrespiratory disease were observed daily on all animals: nasal discharge,ocular discharge, sneezing, retching, anorexia, depression, or coughing.Additionally, tympanic temperatures and nasal/pharyngeal swabs for virusisolation were collected daily.

Clinical observations were reported and tracked daily untilapproximately 50% of the challenged animals exhibited clinical signs ofrespiratory disease. Five days post-challenge, 31 of 59 challengedanimals exhibited one or more of the following clinical signs: nasaldischarge, ocular discharge and/or coughing. Therefore, on Study Day 5,all animals were euthanized and necropsies performed. Lung consolidationscores were recorded and samples for virus isolation collected.

The percent lung consolidation scores indicated that successfuldemonstration of respiratory disease was achieved with this challenge,Percent consolidation across all animals ranged from 60% in 9 animals,to eight animals with 10 to 35% consolidation, 26 animals exhibiting<10% consolidation and 16 animals exhibiting no consolidation (FIG. 2).Numbers of animals with lung consolidation across treatment groups arepresented in Table 4. While numeric differences were observed betweentreatment groups and negative controls, statistically significantdifferences for lung consolidation, virus isolation in lung and tonsiltissues were not detected (Table 5). From lung lavage samples,statistically significant differences (P<=0.05) were detected in thevirus isolation results between negative controls and T02, T07, and T09(Table 6).

TABLE 1 Treatment groups for canine seroconversion study. Group IVP T01Saline - Negative Control T02 Miami 63 - 320 HAI dose - 2% Alum T03Miami 63 - 640 HAI dose - 2% Alum T04 Miami 63 - 320 HAI dose -Quil-A/Cholesterol T05 Miami 63 - 640 HAI dose - Quil-A/Cholesterol T06Ohio 03 - 320 HAI dose - 2% Alum T07 Ohio 03 - 640 HAI dose - 2% AlumT08 Ohio 03 - 640 HAI dose - Quil-A/Cholesterol T09 Ohio 03 - 640 HAIdose - Quil-A/Cholesterol/plus other adjuvants

TABLE 2 Serological Titer Data Summary in dogs vaccinated with equineinfluenza antigen vaccines (36252) HAI titers against CIV Day 21Treatment group Day 0 Geometric Day 35 Group IVP N Actual Mean Min MaxGM Min Max T01 Saline 10 <8 4.0 4.0 4.0 4.0 4.0 4.0 T02 Miami 63 - lowdose - 2% 9 <8 4.0 4.0 4.0 9.3* 4.0 16.0 Alum T03 Miami 63 - high dose -2% 10 <8 4.0 4.0 4.0 12.1* 4.0 128.0 Alum T04 Miami 63 - low dose -Quil- 10 <8 4.0 4.0 4.0 9.2* 4.0 16.0 A/Cholesterol T05 Miami 63 - highdose - 8 <8 4.0 4.0 4.0 7.3 4.0 16.0 Quil-A/Cholesterol T06 Ohio 03 -low dose - 2% 10 <8 17.1* 8.0 64.0 48.5* 32.0 128.0 Alum T07 Ohio 03 -high dose - 2% 10 <8 11.3* 4.0 32.0 55.7* 32.0 128.0 Alum T08 Ohio 03 -high dose - Quil- 10 <8 7.5* 4.0 16.0 18.4* 8.0 32.0 A/Cholesterol T09Ohio 03 - high dose - 10 <8 32.0* 4.0 128.0 84.4* 8.0 256.0 Quil-A/plusother adjuvants *= Significant difference between T01 and marked group(P ≦ 0.05)

TABLE 3 Frequency Distribution of Positive Injection Site Reactions onEach Study Day by Treatment Group Treatment group Positive InjectionSite Reactions Group IVP DO D2 D3 D21 D22 D23 D24 T01 Saline 0 0 0 0 0 00 T02 Miami 63 - low dose - 2% Alum 0 0 0 8/8  7/9 8/9 7/9 T03 Miami63 - high dose - 0 0 0 8/10 10/10 6/9 8/9 2% Alum T04 Miami 63 - lowdose - 0 0 0 0  6/10  6/10  5/10 Quil-A/Cholesterol T05 Miami 63 - highdose - 0 0 0 0 6/9 5/8 5/8 Quil-A/Cholesterol T06 Ohio 03 - low dose -2% Alum 0 0 0 6/10 10/10 7/8 9/9 T07 Ohio 03 - high dose - 2% Alum 0 0 07/10  9/10 10/10  9/10 T08 Ohio 03 - high dose - 0 0 0 0 0 0 0Quil-A/Cholesterol T09 Ohio 03 - high dose - 0 0 0 5/10 10/10 10/10 8/9Quil-A/Cholesterol/plus other adjuvants

TABLE 4 Number of animals per treatment group with positive lungconsolidation scores. IVP T01 T02 T03 T06 T07 T09 No. Positive 8 5 9 7 59 No. Negative 2 4 1 3 5 1

TABLE 5 Virus Isolation from Lung and Tonsil Samples by Treatment GroupVirus Isolated? Yes/No Lung Tonsil No Yes No Yes IVP No. % No. % No. %No. % T01 7 70.0 3 30.0 9 90.0 1 10.0 T02 8 88.9 1 11.1 9 100.0 0 0 T0310 100.0 0 0 10 100 0 0 T06 9 90.0 1 10.0 9 90.0 1 10.0 T07 10 100.0 0 010 100.0 0 0 T09 10 100.0 0 0 10 100.0 0 0

TABLE 6 Virus Isolation from Lung Lavage Samples by Treatment Group.Virus Isolated? Yes/No No Yes IVP Number Percentage Number PercentageT01 2 20.0 8 80.0  T02 7 77.8 2 22.2* T03 5 50.0 5 50.0  T06 7 70.0 330.0* T07 10 100.0 0 0*  T09 10 100.0 0 0*  *Indicates statisticallysignificant difference (p ≦ 0.05) between T01 and treatment group

CONCLUSION OF SPECIFIC DESCRIPTIONS

It should be noted that, as used in this specification and the appendedclaims, singular articles such as “a,” “an,” and “the,” may refer to oneobject or to a plurality of objects unless the context clearly indicatesotherwise. Thus, for example, reference to a composition containing “acompound” may include a single compound or two or more compounds.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patents, patent applications and publications, areincorporated herein by reference in their entirety and for all purposes.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A method of preventing or reducing a disease in a dog caused by asubtype H3 influenza virus, the method comprising administering to thedog a therapeutically effective amount of a vaccine containing at leastone influenza virus, wherein said virus is isolated from a dog, issubtype H3N8, and is killed, wherein said influenza virus consists ofthat deposited with the American Type Culture Collection (ATCC) asPTA-7694, and wherein said vaccine further comprises at least oneantigen selected from the group consisting of canine parainfluenza(CPIV), canine distemper virus (CDV), canine parvovirus (CPV), andcanine adenovirus-2 (CAV-2).
 2. The method of claim 1, wherein saidvaccine further comprises enteric canine coronavirus (CCV).
 3. A methodof preventing or reducing a disease in a dog caused by a subtype H3influenza virus, the method comprising administering to the dog atherapeutically effective amount of a vaccine containing at leastinfluenza virus, wherein said virus is isolated from a dog. is subtypeH3N8, and is killed, wherein said influenza virus consists of thatdeposited with the American Type Culture Collection (ATCC) as PTA-7694,and wherein said vaccine further comprises at least one antigen selectedfrom the group consisting of Leptospira canicola, Leptospiragrippotyphosa, Leptospira icterohaemorrhagiae, Leptospira pomona, andLeptospira bratislava.
 4. The method of claim 1, wherein said vaccinefurther comprises at least one antigen selected from the groupconsisting of Leptospira canicola, Leptospira grippotyphosa, Leptospiraicterohaemorrhagiae, Leptospira pomona, and Leptospira bratislava. 5.The method of claim 2, wherein said vaccine further comprises at leastone antigen selected from the group consisting of Leptospira canicola,Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, Leptospirapomona, and Leptospira bratislava.